Understanding the complex regulation of beta-globin genes is critically important to design therapeutic approaches to beta-thalassemia and sickle cell disease. We have described an erythroid-specific protein complex containing LDB1/GATA-1/TAL1/LMO2 (LDB1 complex). that is directly involved in activation of globin gene expression. Through dimerization of LDB1, the complex mediates long-range interaction between the beta-globin LCR enhancer and globin gene promoters. Genome-wide studies show that the LDB1 complex regulates erythroid enhancers, more generally. However, some gene targets of LDB1 enhancers are not occupied by LDB1, raising the question of additional mechanisms for looping by LDB1. We find that a novel LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Genome-wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression. Cohesin is a frequent partner of CTCF genome-wide and has been reported to participate directly in enhancer-gene contacts in embryonic stem cells. However, we find that cohesin is almost completely absent from LDB1-regulated erythroid enhancer-gene pairs, whether or not they involved CTCF. Enhancer-gene pairs are primarily located within topologically associated domain, or TADs, that are thought to be looped domains created by interactions between CTCF sites at the TAD borders. Do such indirect loops favor the internal enhancer-gene interations or is the reverse true? To address this issue in vivo, we used CRISPR/Cas9 technology to delete CTCF sites flanking the beta-globin locus over 1 Mb of chromosome 11 in human erythroid K562 and non-erythroid 293T cells. Bi-allelic deletions were validated by PCR. None of the deletions affected CTCF levels in cells according to Western blots and each deletion resulted in loss of CTCF binding uniquely at that site according to ChIP analysis. Histone modifications in the vicinity of each deletion are being analyzed by CHIP-seq. Globin gene expression and expression of Trim genes adjacent to the globin locus were affected in different ways by deletion of individual CTCF sites. To further understand how CTCF binding site deletion alters chromosome architecture and local gene expression, we are performing CTCF-capture Hi-C. Transcriptional factor ETO2 can participate in the LDB1 complex and this confers negative function on the complex but the mechanisms involved are not known. ETO2 can recruit HDACs to target genes through its dimerization domain and zinc finger region, which may underlie the repressor function. We knocked out ETO2 in K562 cells using CRISPR-Cas9 technology and performed RNA-seq to determine differentially regulated genes. Embryonic and fetal gamma globin genes as well as erythroid specific transcription factors were up-regulated in the absence of ETO2. In addition, loss of ETO2 caused significant decrease of HDAC1 and LSD1 occupancy and significant increase of H3K27ac in the beta-globin LCR region and increased H3K9ac and H3K4me2 at the gamma-globin gene promoter. Furthermore, ETO2 null cells showed higher interaction frequency between the LCR and gamma-globin promoter than WT cells and increased loading of LDB1 complex, which mediates loop formation, at the LCR. We next expressed ETO2 variant proteins missing either the TAF110, dimerization and zinc finger domains in the ETO2 null background. In summary, we found that all three domains are required for ETO2 to function as a transcriptional repressor in gamma globin gene regulation, suggesting potential therapeutic targets for sickle cell disease and beta-thalassemia.